Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. We investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution.

Is vision necessary for the development of the categorical organization of the Ventral Occipito-Temporal Cortex (VOTC)? We used fMRI to characterize VOTC responses to eight categories presented acoustically in sighted and early blind individuals, and visually in a separate sighted group. We observed that VOTC reliably encodes sound categories in sighted and blind people using a representational structure and connectivity partially similar to the one found in vision. Sound categories were, however, more reliably encoded in the blind than the sighted group, using a representational format closer to the one found in vision. Crucially, VOTC in blind represents the categorical membership of sounds rather than their acoustic features. Our results suggest that sounds trigger categorical responses in the VOTC of congenitally blind and sighted people that partially match the topography and functional profile of the visual response, despite qualitative nuances in the categorical organization of VOTC between modalities and groups.

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex (G4-DNA) structures. G4-DNA regulates replication and transcription, at least in cancer cells. Here, we demonstrate that, in neurons, pharmacologically stabilizing G4-DNA with G4 ligands strongly downregulates the Atg7 gene. Atg7 is a critical gene for the initiation of autophagy that exhibits decreased transcription with aging. Using an in vitro assay, we show that a putative G-quadruplex-forming sequence (PQFS) in the first intron of the Atg7 gene folds into a G4. An antibody specific to G4-DNA and the G4-DNA-binding protein PC4 bind to the Atg7 PQFS. Mice treated with a G4 stabilizer develop memory deficits. Brain samples from aged mice contain G4-DNA structures that are absent in brain samples from young mice. Overexpressing the G4-DNA helicase Pif1 in neurons exposed to the G4 stabilizer improves phenotypes associated with G4-DNA stabilization. Our findings indicate that G4-DNA is a novel pathway for regulating autophagy in neurons.

Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate αSyn aggregation and disease in the gut and the brain.

The signal regulated transcription factors (SRTFs) control the ultimate transcriptional output of signaling pathways. Here, we examined a family of FGF-induced SRTFs - Etv1, Etv 4, and Etv 5 - in murine lens development. Contrary to FGF receptor mutants that displayed loss of ERK signaling and defective cell differentiation, Etv deficiency augmented ERK phosphorylation without disrupting the normal lens fiber gene expression. Instead, the transitional zone for lens differentiation was shifted anteriorly as a result of reduced Jag1-Notch signaling. We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression, which is necessary for the nuclei clearance in mature lens. These results revealed the functional divergence between Etv and FGF in lens development, demonstrating that these SRTFs can operate outside the confine of their upstream signaling.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)'s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.

Despite advances in methods to detect protein synthesis, it has not been possible to measure endogenous protein synthesis levels in vivo in an entire vertebrate brain. We developed a transgenic zebrafish line that allows for cell-type-specific labeling and imaging of nascent proteins in the entire animal. By replacing leucine with glycine in the zebrafish MetRS-binding pocket (MetRS-L270G), we enabled the cell-type-specific incorporation of the azide-bearing non-canonical-amino-acid azidonorleucine (ANL) during protein synthesis. Newly synthesized proteins were then labeled via 'click chemistry'. Using a Gal4-UAS-ELAV3 line to express MetRS-L270G in neurons, we measured protein synthesis intensities across the entire nervous system. We visualized endogenous protein synthesis and demonstrated that seizure-induced neural activity results in enhanced translation levels in neurons. This method allows for robust analysis of endogenous protein synthesis in a cell-type-specific manner, in vivo at single-cell resolution.

Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na+ channels (Navs). It is known that prokaryotic Navs can obtain Ca2+ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca2+ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca2+ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs. These findings provide new insight into the Ca2+ selectivity mechanism that is conserved from prokaryotes to eukaryotes.

Phylogenetic relationships among extinct hominoids (apes and humans) are controversial due to pervasive homoplasy and the incompleteness of the fossil record. The bony labyrinth might contribute to this debate, as it displays strong phylogenetic signal among other mammals. However, the potential of the vestibular apparatus for phylogenetic reconstruction among fossil apes remains understudied. Here we test and quantify the phylogenetic signal embedded in the vestibular morphology of extant anthropoids (monkeys, apes and humans) and two extinct apes (Oreopithecus and Australopithecus) as captured by a deformation-based 3D geometric morphometric analysis. We also reconstruct the ancestral morphology of various hominoid clades based on phylogenetically-informed maximum likelihood methods. Besides revealing strong phylogenetic signal in the vestibule and enabling the proposal of potential synapomorphies for various hominoid clades, our results confirm the relevance of vestibular morphology for addressing the controversial phylogenetic relationships of fossil apes.

SLAMF6 is a homotypic receptor of the Ig-superfamily whose exact role in immune modulation has remained elusive. Its constitutive expression on resting and activated T cells precludes it from being a bona fide exhaustion marker. By breeding Pmel-1 mice with SLAMF6 -/- mice, we generated donors for T cells lacking SLAMF6 and expressing a transgenic TCR for gp100-melanoma antigen. Activated Pmel-1xSLAMF6 -/- CD8+ T cells displayed improved polyfunctionality and strong tumor cytolysis. T-bet was the dominant transcription factor in Pmel-1 x SLAMF6 -/- cells, and upon activation, they acquired an effector-memory phenotype. Adoptive transfer of Pmel-1 x SLAMF6 -/- T cells to melanoma-bearing mice resulted in lasting tumor regression in contrast to temporary responses achieved with Pmel-1 T cells. LAG-3 expression was elevated in the SLAMF6 -/- cells, and the addition of the LAG-3-blocking antibody to the adoptive transfer protocol improved the SLAMF6 -/- T cells and expedited the antitumor response even further. The results from this study support the notion that SLAMF6 is an inhibitory immune receptor whose absence enables powerful CD8+ T cells to eradicate tumors.

PD-1 blockade therapy has revolutionized cancer treatments. However, a substantial population of patients is unresponsive. To rescue unresponsive patients, the mechanism of unresponsiveness to PD-1 blockade therapy must be elucidated. Using a 'bilateral tumor model' where responsive and unresponsive tumors were inoculated into different sides of the mouse belly, we demonstrated that unresponsive tumors can be categorized into two groups: with and without systemic immunosuppressive property (SIP). The SIP-positive tumors released uncharacterized, non-proteinaceous small molecules that inhibited mitochondrial activation and T cell proliferation. By contrast, the SIP-negative B16 tumor escaped from immunity by losing MHC class I expression. Unresponsiveness of SIP-positive tumors was partially overcome by improving the mitochondrial function with a mitochondrial activator; this was not successful for B16, which employs immune ignorance. These results demonstrated that the 'bilateral tumor model' was useful for stratifying tumors to investigate the mechanism of unresponsiveness and develop a strategy for proper combination therapy.

A vaccine protective against diverse HCV variants is needed to control the HCV epidemic. Structures of E2 complexes with front layer-specific broadly neutralizing antibodies (bNAbs) isolated from HCV-infected individuals, revealed a disulfide bond-containing CDRH3 that adopts straight (individuals who clear infection) or bent (individuals with chronic infection) conformation. To investigate whether a straight versus bent disulfide bond-containing CDRH3 is specific to particular HCV-infected individuals, we solved a crystal structure of the HCV E2 ectodomain in complex with AR3X, a bNAb with an unusually long CDRH2 that was isolated from the chronically-infected individual from whom the bent CDRH3 bNAbs were derived. The structure revealed that AR3X utilizes both its ultralong CDRH2 and a disulfide motif-containing straight CDRH3 to recognize the E2 front layer. These results demonstrate that both the straight and bent CDRH3 classes of HCV bNAb can be elicited in a single individual, revealing a structural plasticity of VH1-69-derived bNAbs.

Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae. The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HAts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways.

Melatonin receptors MT1 and MT2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

Skin vasculature cross-talking with hair follicle stem cells (HFSCs) is poorly understood. Skin vasculature undergoes dramatic remodeling during adult mouse hair cycle. Specifically, a horizontal plexus under the secondary hair germ (HPuHG) transiently neighbors the HFSC activation zone during the quiescence phase (telogen). Increased density of HPuHG can be induced by reciprocal mutations in the epithelium (Runx1) and endothelium (Alk1) in adult mice, and is accompanied by prolonged HFSC quiescence and by delayed entry and progression into the hair growth phase (anagen). Suggestively, skin vasculature produces BMP4, a well-established HFSC quiescence-inducing factor, thus contributing to a proliferation-inhibitory environment near the HFSC. Conversely, the HFSC activator Runx1 regulates secreted proteins with previously demonstrated roles in vasculature remodeling. We suggest a working model in which coordinated remodeling and molecular cross-talking of the adult epithelial and endothelial skin compartments modulate timing of HFSC activation from quiescence for proper tissue homeostasis of adult skin.